Method of treating anemia caused by ribavirin treatment of hepatitis C using erythropoietin alpha

ABSTRACT

Claimed and disclosed is a new use for a previously approved drug: erythropoietin. The present invention teaches using Erythropoetin to treat anemia caused by the combined treatment of Ribavirin and alpha-interferon. Erythropoetin has previously been approved for the treatment of anemia caused by cancer chemotherapy, renal failure and HIV. It has not been used for anemia caused by ribavirin. Ribavirin is part of a two-drug regimen now used to treat hepatitis C along with alpha interferon. The principal side effect of ribavirin is a hemolytic anemia. In the past, management of that anemia was done by dose reduction of the ribavirin, sometimes resulting in reversal of part of the anemia. It has become particularly important in light of new data, to maximize the dose of ribavirin given to persons undergoing treatment for hepatitis C to ensure a successful eradication of hepatitis C.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/976,723, filed on Oct. 28, 2004, which is a divisional of U.S.application Ser. No. 09/862,404, filed on May 21, 2001, issued as U.S.Pat. No. 6,833,351.

FIELD OF THE INVENTION

The present invention is directed to a new use for Erythropoetin(“EPO”), such as EPO alpha, for treating hepatitis C and/or anemiacaused by hepatitis C treatment. Accordingly, the invention involvesusing EPO with hepatitis C treatment, such as Ribavirin (“RBV”) and/orinterferon such as alpha-interferon (“α-IFN” or “IFN”); and thus, theinvention pertains to methods involving administration of EPO, RBV andα-IFN, or EPO and RBV, and compositions and kits containing EPO, RBV andα-IFN or EPO and RBV.

Various documents are cited herein, e.g., in the text and/or in areference section. There is no admission that any of the variousdocuments cited in this text are prior art as to the present invention.Any document having as an author or inventor person or persons named asan inventor herein is a document that is not by another as to theinventive entity herein. All documents cited in this text (“herein citeddocuments”) and all documents cited or referenced in herein citeddocuments are hereby incorporated herein by reference. Allspecifications, manufacturer's data sheets, and the like for productsreferenced herein, and all documents cited therein, are herebyincorporated herein by reference.

BACKGROUND OF THE INVENTION

Erythropoietin (“EPO”) is one of the red blood cell stimulating factorsin the human body. Recombinant technology has made manufacture of thisstimulating factor colony possible and its use in treating anemia causedby cancer chemotherapy, acquired immune deficiency syndrome (“AIDS”) andrenal failure. The recombinant product has been shown to be biologicallyidentical to human erythropoietin.

A normal response to anemia in humans is the release of EPO and acorresponding rise in the hemoglobin level. There can be either aninadequate production of EPO or a lack of response to EPO by the bonemarrow. When ribavirin is administered by itself, it gets pumped intored blood cells and, once inside, gets phosphorylated. After it isphosphorylated, the ribavirin cannot get out of the red blood cell andits concentration builds up until the red blood cell bursts (hemolysis).This anemia can be severe and even life threatening, particularly inpeople with heart disease. The normal response to a hemolytic anemia isthe release of EPO which stimulates the bone marrow to produce more redblood cells, and immature red blood cells (reticulocytes) will bediscovered in the peripheral blood smear. When interferon isadministered with ribavirin, however, it blocks the normal response ofthe bone marrow to respond to the anemia and the reticulocyte responseis blunted. Administration of exogenous EPO such as Epoetin alpha canovercome this response and cause the bone marrow to produce more redblood cells, overcoming the inhibitory effect of the interferon.

Recently, great importance has been placed on using ribavirin toeradicate hepatitis C. Hepatitis C, according to the Centers for DiseaseControl (“CDC”), afflicts over 3.9 million people in the United Statestoday. Cirrhosis and liver failure caused by hepatitis C are the leadingcauses of liver transplant in the U.S. today. It is also an importantglobal problem with as many as 50 million people afflicted withhepatitis C worldwide. The combination of interferon and ribavirin hasbeen an approved treatment for hepatitis C since 1998. The addition ofribavirin has more than doubled the effect of the treatment to thehepatitis C virus. Clinical data from recent licensing trials shows thatpatients who receive at least 10.6 mg/kg of ribavirin have a sustainedvirologic response rate of over 40%, whereas those receiving less thanthat amount have a SVR (response) rate of only 28%. The maximum benefitis gained from a dose of ribavirin that is about 13 mg/kg. Such highdoses of ribavirin cause a substantial increase of almost 50% in theincidence of anemia. The normal management of this would be to reducethe ribavirin dose and thereby reduce the efficacy of theinterferon/ribavirin combination therapy. However, this would defeat thepurpose of the hepatitis C treatment.

With respect to treating anemia caused by hepatitis C treatment inconjunction with ribavirin, reference is made to the following:

Albrecht, U.S. Pat. No. 6,172,046 B1, relates to a method of treating apatient having chronic hepatitis C infection. To eradicate detectablehepatitis C virus RNA, a combination therapy using a therapeuticallyeffective amount of ribavirin and a therapeutically effective amount ofinterferon-alpha for a time period of from 20 up to 80 weeks isdisclosed.

Draper, U.S. Pat. No. 5,610,054, relates to an enzymatic RNA moleculewhich cleaves RNA of the hepatitis C virus. Draper, U.S. Pat. No.5,869,253, relates to an enzymatic RNA molecule which cleaves RNAencoding hepatitis C virus (“HCV”) RNA, wherein the enzymatic RNAmolecule comprises a substrate binding site and a nucleotide sequencewithin or surrounding the substrate binding site wherein the nucleotidesequence imparts to the enzymatic RNA molecule activity for the cleavageof the HCV RNA. Draper, U.S. Pat. No. 6,132,966, relates to an enzymaticnucleic acid molecule which inhibits hepatitis C virus replication.

Ise et al., U.S. Pat. No. 5,399,551, relates to an enhancer forantianemia effect of erythropoietin using a spherical carbon as anactive ingredient in an amount effective for treating anemia incombination with erythropoietin. The enhancer is orally administered.The antianemia effect of erythropoietin can be enhanced, the dosage oferythropoietin can be decreased, and side effects from erythropoietincan be reduced. The invention further discloses a method of augmentingthe anti-anemia effect of Erythropoetin by administering to a patient aneffective amount of a spherical carbon in combination with a portion ofan effective amount of Erythropoetin for treating anemia.

Population Pharmacokinetic and Pharmacodynamic Analysis of Ribavirin inPatients with Chronic Hepatitis C, by J. Frank Jen, Paul Glue, SamirGupta, Demetrius Zambas and Gerald Hajian (Therapeutic Drug Monitoring,Vol. 22, No. 3, 2000) (“Population”), reported that

-   -   “Although anemia is a well-established adverse effect of        ribavirin therapy, the association between drug concentrations        and extent of anemia has not been thoroughly investigated.        Earlier reports (22, 23) found that greater mean falls in        hemoglobin were associated with higher daily ribavirin doses.    -   . . . Despite the mean trends the variability of these data was        high, suggesting that it would not be possible to establish a        concentration range below which hemolysis would not occur, or        above which anemia was inevitable. From a practical perspective,        the most appropriate method of dealing with treatment related        anemia would appear to be through dose reduction of ribavirin,        although this analysis indicates that this should be based on        individual hemotologic responses to ribavirin rather than based        on serum ribavirin concentrations.”

Thus, there is teaching in the art to reduce RBV—reduce the HCVtreatment—to address anemia. Teachings such as this teach away fromaddressing anemia by other means and therefore teach away from thepresent invention.

Poduslo et al., U.S. Pat. No. 5,604,198, relates to a method ofenhancing an ability of a neurologically active compound to penetratethe blood nerve barrier (“BNB”) or blood brain barrier (“BBB”), byadministering a conjugate comprising the neurologically active compoundlinked to a carrier molecule that has been shown to have a substantialpermeability coefficient across the BNB and BBB. Poduslo et al., U.S.Pat. No. 5,670,477, relates to a method of enhancing the ability of aneurologically active compound to penetrate the blood nerve barrier(“BNB”) or blood brain barrier (“BBB”) comprising parenterallyadministering to a mammal in need of treatment with the neurologicallyactive compound, a conjugate consisting of an effective amount of theneurologically active compound linked to a polyamine having asubstantial permeability coefficient across the BNB or BBB.

Strickland, U.S. Pat. No. 5,661,125, relates to stable compositions oferythropoietin that contain an antimicrobial preservative therebyproviding a multi-dose formulation. Preservatives useful in thepharmaceutical compositions of the invention include benzyl alcohol,parabens, phenol and mixtures thereof. Other additives, includingbuffers, may be included in the composition.

Tam, U.S. Pat. No. 6,063,772, relates to administering ribavirin to apatient in a dosage range which is effective to modulate lymphokineexpression in activated T cells. In particular, ribavirin is used tosuppress Th2-mediated T cell responses and promote Th1-mediated T cellresponse. Instead of administering ribavirin in its well-recognized roleas an anti-viral agent, ribavirin is thus used in the treatment ofimbalances in lymphokine expression. Such imbalances may be found to beconcomitants of allergic atopic disorders, such as allergic asthma andatopic dermatitis, helminthes infection and leishmaniasis, and variousprimary and secondary immunodeficiencies, which may or may not also beassociated with viral infection.

Interferon (IFN) and Ribavirin (RBV) Therapy for Hepatitis C (HCV) inHIV-Coinfected Patients, 12 Month Follow-Up, 7^(th) Conference onRetroviruses and Opportunistic Infections, at the Birth of a Century,Research Toward Ending AIDS, Program and Abstracts, Jan. 30-Feb. 2,2000, by K. Weisz, D. Goldman, A. Talal, M. Malicdem and D. Dieterich,reported that (1) IFN alone does not adversely affect HIV RNA, and canhave little effect on HCV RNA; (2) IFN and RBV have little effect on HIVRNA clinically, and have a sizeable effect on HCV RNA; (3) anemia, aside effect of RBV, can be successfully treated with erythropoietin.However, this study fails to teach or suggest the present invention asit did not involve co-administered or concomitant administration asherein; and, because the study concerned and HIV positive patients, notHCV patients who are HIV negative or immunocompetent, as there is aprejudice in the art against employing EPO in immunocompetentindividuals because EPO administration can give rise to anti-EPOantibodies. (However, methods, kits and compositions of the inventionare not necessarily excluded from being employed with respect toimmunocompromised individuals.)

Yatvin et al., U.S. Pat. No. 5,543,390, relates to a method and areagent for delivering biologically active compounds to phagocyticmammalian cells. The patent also relates to an uptake of suchbiologically active compounds by phagocytic cells and delivery of suchcompounds to specific sites intracellularly. The invention moreparticularly relates to methods of facilitating the entry ofantimicrobial drugs and other agents into phagocytic cells and fortargeting such compounds to specific organelles within the cell. The'390 patent further relates to compositions of matter and pharmaceuticalembodiments of such compositions comprising conjugates of suchantimicrobial drugs and agents covalently linked to particulate carriersgenerally termed microparticles. In particular embodiments, theantimicrobial drug is covalently linked to a microparticle via anorganic linker molecule which is the target of a microorganism-specificprotein having enzymatic activity. The 390 patent also provides celltargeting of drugs wherein the targeted drug is only released in cellsinfected with a particular microorganism. Alternative embodiments ofsuch specific drug delivery compositions also contain polar lipidcarrier molecules effective in achieving intracellular organelletargeting in infected phagocytic mammalian cells. Particular embodimentsof such conjugates comprise antimicrobial drugs covalently linked bothto a microparticle via an organic linker molecule and to a polar lipidcompound, to facilitate targeting of such drugs to particularsubcellular organelles within the cell. Also provided are porousmicroparticles impregnated with antimicrobial drugs and agents whereinthe surface or outside extent of the microparticle is covered with adegradable coating that is specifically degraded within an infectedphagocytic mammalian cell. Methods of inhibiting, attenuating,arresting, combating and overcoming microbial infection of phagocyticmammalian cells in vivo and in vitro are also provided. While technologyin the 390 patent may be useful in the practice of the herein invention,the 390 patent fails to teach or suggest the present invention.

Yatvin et al., U.S. Pat. No. 5,543,391, relates to a composition havinga biologically-active compound that is an antimicrobial drug, a porousmicroparticle, and an organic coating material, wherein thebiologically-active compound is impregnated within the porousmicroparticle, and the microparticle is coated with the organic coatingmoiety, and wherein the organic coating material comprises a compoundthat is specifically degraded inside a phagocytic mammalian cellinfected with a microorganism to allow release of thebiologically-active compound within the infected cell.

Foregoing patents and/or other background information discussed mayinvolve either an EPO composition or an EPO enhancer. However, none ofthe patents foregoing patents and/or background information discloses orsuggests using EPO as an anti-anemia treatment in conjunction with theribavirin-interferon-alpha treatment for HIV negative or immunocompetenthepatitis C patients and/or in a co-administration regimen and/or via akit, as herein.

And, in this regard, it is again mentioned that administration of EPOcan give rise to anti-EPO antibodies, inter alia, such that there may bea prejudice against administration of EPO to immuno-competent (e.g., HIVnegative) individuals.

Thus, it is believed that heretofore the present invention has not beentaught or suggested.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a new use ofErythropoetin (“EPO”) to treat an anti-anemia condition caused by thecombination treatment of ribavirin and interferon-alpha. It is anotherobject of the present invention to provide a method of administering EPOto patients with ribavirin-induced anemia to counteract a hemolyticeffect and to allow patients to take the maximum effective dosage ofribavirin and interferon-alpha necessary to eradicate hepatitis C.

It is and has been surprisingly found that EPO works the same in bothHIV positive and HIV negative patients with HCV.

In this text, the term “comprising”, “comprise”, “comprises” and otherforms of “comprise” can have the meaning described to these terms inU.S. Patent Law and can mean “including”, “include”, “includes” andother forms of “include”.

Co-administering is administration at the same time or at about the sametime, i.e., via a composition or kit containing all of EPO,interferon-alpha and ribavirin; for instance, co-administering caninvolve a sequential administration of RBV, EPO and IFN e.g., via avisit to a healthcare provider, or via patient self administration.

More in particular, RBV typically is provided and administered in solid,e.g., capsule or tablet form or in the form of an inhaler, with thetablet or capsule being usually employed with respect to HCV. Patientsusually take 2 to 3 tablets or capsules of RBV/day (e.g., Rebetol, suchas 800 or 1000-1200 mg/daily). IFN is provided in an injectable form, asis EPO. For instance, Intron A (IFN) injection in a 3 MIU dose can beadministered three times weekly; and PEG-IntronNT (pegylated IFN)injection in a 0.5 or 1.5 mcg/kg dose can be administered once weekly(QW). Combinations of RBV and IFN can include: PEG-IntronNT injection1.5 mcg/kg once weekly (QW) plus Rebetol capsules 800 mg/daily for 48weeks (PEG 1.5/R); PEG-Intron 1.5 mcg/kg QW plus Rebetol 1000-1200mg/daily for four weeks followed by PEG-Intron 0.5 mcg/kg QW plusRebetol 1000-1200 mg/daily for 44 weeks (Peg 0.5/R); or Intron Ainjection 3 MIU/three times weekly plus Rebetol Capsules 1000-1200mg/daily for 48 weeks (Rebetron). Patients can self-administer EPO, RBVand IFN.

The invention envisages kits that contain EPO, RBV and IFN, such thatthe patient can administer the EPO, RBV and IFN; e.g., RBV orally twicedaily (e.g. once in morning, once in evening or three times dailyincluding a morning dose), IFN once weekly or three times weekly viainjection, and EPO at least once weekly via injection. The patient cantherefore obtain a one-week, two-week, three-week, monthly, bi-monthly,three-month, four-month, five-month, six-month, seven-month,eight-month, nine-month, ten-month, eleven-month, or annual supply ofEPO, RBV and IFN, via a kit of the invention.

The kit thus advantageously contains EPO e.g., for injection, IFN e.g.,for injection, suitable devices for administration via injection (e.g.,syringes) if components are supplied for administration via injection,RBV e.g., tablets or capsules, and optionally (but advantageously)instructions for administration/use. The patient can therefore each dayself-administer or be administered the RBV (e.g., orally),advantageously with a first dose in the morning or upon awakening. Onselected day or day(s), such as in the morning or upon awakening, withthe first dose of RBV, the patient can then also administer IFN. And, onselected day or day(s), such as in the morning or upon awakening, alsowith a first dose of RBV, the patient can additionally administer theEPO.

Thus, the patient can be having a co-administration of EPO and RBV.

Moreover, at least once weekly, the patient can be havingco-administration of RBV, IFN and EPO, advantageously at the same time,e.g., via oral administration of RBV and sequential injections of IFNand EPO.

The patient self administrations can, of course, be performed by ahealthcare provider.

Even more advantageous, the EPO and IFN can be provided for admixtureand/or in an admixed state, so that the EPO and IFN administration isvia one injection of a combination of EPO and IFN. Thus, when the EPOand IFN are to be co-administered, it may be possible to admix them fora single injection containing both. However, an injection for each ofthe EPO and IFN and oral administration of RBV are presently preferred.

A preferred embodiment involves a kit containing EPO for once weeklyadministration by injection, IFN for once weekly administration byinjection, and RBV for twice daily oral administration. The kit cancontain instructions for administration. The kit can also containdevices for administration via injection. Again, the kit can contain an“N” week, wherein “N” is an integer advantageously from 1-52 (e.g., oneweek, or two week, or three week, or four week, or five week, or sixweek, or seven week, or eight week, or nine week, or ten week, or elevenweek, or 12 week or 13 week or 14 week etc. up to 52 week) supply ofEPO, IFN and RBV.

Accordingly, the invention provides methods for administering RBV andEPO or RBV, EPO and IFN for treatment of HCV and/or anemia caused byHCV, as well as compositions containing EPO and IFN, and kits containingRBV and EPO, or, RBV, EPO and IFN, with such kits optionally containinginstructions for administration and/or devices for administration, andthe EPO and IFN, if together in the kit, can be in forms so that whenco-administered they can be admixed prior thereto and/or they are in anadmixed form for co-administration.

In instances where the HCV can be treated by RBV without IFN,embodiments can involve the foregoing, with the IFN omitted.

The invention further comprehends methods for co-administering RBV andEPO or RBV, EPO and IFN, as well as compositions containing EPO and IFN,or RBV and EPO, or RBV, IFN and EPO.

These and other objects and embodiments of the invention are providedin, or are obvious from, the following detailed description of theinvention.

BRIEF DESCRIPTION OF THE DRAWING(S)

FIG. 1 shows a study design and results thereof for administering EPOwith RBV and/or IFN-α.

FIG. 2 shows a collection of plots of hemoglobin levels by time for 18EPO-treated patients. FIG. 2A represents Subject #2; FIG. 2B representsSubject #3; FIG. 2C represents Subject #4; FIG. 2D represents Subject#5; FIG. 2E represents Subject #6; FIG. 2F represents Subject #7; FIG.2G represents Subject #8; FIG. 2H represents Subject #11; FIG. 2Irepresents Subject #13; FIG. 2J represents Subject #14; FIG. 2Krepresents Subject #15; FIG. 2L represents Subject #16; FIG. 2Mrepresents Subject #18; FIG. 2N represents Subject #19; FIG. 2Orepresents Subject #20; FIG. 2P represents Subject #21; FIG. 2Qrepresents Subject #22; and FIG. 2R represents Subject #23.

DETAILED DESCRIPTION OF THE INVENTION

The present invention claims and discloses a new use for erythropoietinin treating ribavirin-interferon-alpha induced anemia. The presentinvention can be administered to humans as well as animals infected withHepatitis C virus or HIV or HIV and HCV or who are suffering fromanemia. The Erythropoetin can be administered in a liquid preparationand/or administered as a vector for in vivo expression. Specifically,the present invention also evaluates the clinical benefit oferythropoietin in ribavirin/interferon-induced anemia.

The combination of ribavirin (RBV) and interferon-alpha (IFN-α) isstandard treatment for hepatitis C (HCV)-infected patients. Anemia(hemoglobin <10 g/dl), severe enough to warrant dose reductions orcessation of therapy, occurs in up to 10% of individuals prescribedthese medications, but a drop in hemoglobin of >3 grams/dl occurs in 54%of people treated with RBV and IFN-α.

Nearly four million individuals in the United States, approximately twopercent of the population, are infected with the hepatitis C virus. Ofthe 30,000 acute cases annually, as many as 85% will progress to chronicinfection. With 8,000 to 10,000 deaths each year attributed to HCV inthe U.S, the infection has become the primary indication for hepatictransplantation. In HIV seropositive patients, as many as 40% of thenearly 1 million HIV seropositive patients have HCV.

Biochemical (normalization of alanine aminotransferase), virologic (lossof serum HCV RNA), and histologic (improvement in hepatic histology)indicators are used to determine a response to therapy for HCV.

Monotherapy with IFN-α 3 million units three times per week (TIW)results in a biochemical end-of-treatment response in 35 to 50% ofsubjects and a sustained response in 10 to 35% of subjects. Recently thecombination of IFN-α and RBV, a synthetic nucleoside analog withantiviral activity, was approved by the U.S. Food and DrugAdministration for the treatment of HCV. The sustained response rate ismore than twice as high among subjects treated with the combination ofIFN-α/RBV than among those treated with IFN-α alone.

Anemia is a major side effect of IFN-α/RBV combined therapy,necessitating dosage reduction in more than 10% of treated individuals.Although the primary mechanism of anemia in patients who receive thecombination of IFN-α and RBV is thought to be hemolysis, othermechanisms, such as the down-regulation of the erythropoietin receptor,may contribute. In response to hypoxia, erythropoietin stimulates theproduction, maturation, and release of erythrocytes from the bonemarrow. In cases of endogenous erythropoietin deficiency or aninadequate hypoxic response, recombinant human erythropoietin (r-HuEPO,Epoetin alpha) improves anemia associated with HIV-infection, chronicdiseases, and malignancies. Similarly, anemia induced by severalmedications including zidovudine, a synthetic nucleoside analog similarto RBV, is improved by Epoetin alpha. Therefore, the present inventionevaluated the benefit of Epoetin alpha in HCV-infected individuals whobecame anemic after the initiation of the IFN-α/RBV combined treatment.

The combination of IFN-α/RBV for the treatment of HCV infection morethan doubles the sustained response rate when compared to IFN-αmonotherapy. Anemia, however, is a dose-limiting side effect of thecombination therapy in a significant patient population, e.g., more than10% of individuals who receive the medications. The inventor of thepresent invention has shown that Epoetin alpha increases hemoglobinlevels in HCV-infected patients who become anemic after IFN-α/RBVtherapy to a level equivalent to that of patients who do not developanemia.

Hepatitis C is the newest opportunistic infection in HIV patients asdeclared by the U.S. Public Health Service in 1999. It progresses tocirrhosis and death much more rapidly in patients with HIV and is theleading cause of death in many HIV clinics. Since the era of highlyactive antiretroviral therapy began in 1995, HIV patients are livinglonger and suffering more ill effects of their hepatitis C infection. Ithas become vital therefore to treat hepatitis C in patients with HIV.One of the early complications of HIV and its treatment was anemia.Erythropoietin has been used to successfully treat HIV-related anemia.Indeed, anemia in HIV patients has been shown to have a negative effecton survival. Thus, many physicians were reluctant to use interferon andribavirin in HIV-infected patients because of their fear of anemia andbecause of concern about ribavirin inhibiting HIV medication.

Although anemia associated with IFN-α/RBV therapy for HCV infection isusually attributed to hemolysis, other mechanisms, such as anemia ofchronic disease (ACD), may contribute. ACD is common in patients withinfectious, inflammatory, and neoplastic disorders, and it is thought toresult from three processes: a moderate shortening of erythrocytesurvival, an inability of the bone marrow to respond to the resultingdemand for increased erythrocyte production, and the impairedmobilization of iron stores in the reticuloendothelial system.

Reduced responsiveness of the bone marrow to erythropoietin has beendescribed in association with malignancy, rheumatoid arthritis, and HIVinfection. In the setting of a demand for increased erythrocyteproduction, the impaired ability of the bone marrow to respond toerythropoietin results in reduced erythrocyte production and anemia.Several proinflammatory cytokines, including tumor necrosis factor-α(TNF-α), interleukin-1a (IL-1a), interleukin-6 (IL-6), and interferon-γ(IFN-γ), have been associated with ACD and a down-regulation inendogenous erythropoietin levels. Chronic infection with HCV isassociated with an increase in proinflammatory cytokines particularlyIFN-γ, TNF-α, and IL-2. Intrahepatic proinflammatory cytokine mRNA isincreased in HCV-infected individuals compared to uninfected controlsand further increases are associated with worsening hepatic injury.

The association between proinflammatory cytokines and ACD and theincrease in proinflammatory cytokines observed in HCV-infectedindividuals raises the possibility that these cytokines may contributeto reduced bone marrow responsiveness to erythropoietin. The results ofthis study raise the possibility that two separate processes mayunderlie the hemoglobin reduction in HCV-infected individuals whodevelop anemia after initiation of IFN-α/RBV treatment, a hemolyticcomponent and a biochemical component. RBV may result in an increase inerythrocyte hemolysis while an increase in proinflammatory cytokines mayresult in decreased responsiveness of the bone marrow to erythropoietin.Furthermore, the reduced bone marrow responsiveness to erythropoietinmay only become apparent after the onset of erythrocyte hemolysissecondary to IFN-α/RBV.

Epoetin alpha significantly increases hemoglobin levels in severaldisorders characterized by decreased bone marrow responsiveness toerythropoietin, including rheumatoid arthritis, cancer, and HIVinfection. Epoetin alpha increases the number of reticulocytes releasedfrom the bone marrow into the circulation and reduces apoptosis amonglate erythroid precursors, thereby increasing hemoglobin levels. It mayalso compensate for the inhibitory effect of proinflammatory cytokineson erythropoiesis and, thus, may be an efficacious therapy forIFN-α/RBV-associated anemia in HCV-infected individuals.

Both IFN-α and RBV are capable of directly inhibiting erythropoiesisthrough mechanisms that are known to be sensitive to the action ofEpoetin alpha. For example, interferons reduce the growth rate of normalhematopoietic cells, thereby inhibiting the proliferation of erythroidprogenitor cells. In addition, IFN-α diminishes erythropoietinproduction in hepatocyte cultures. RBV is a synthetic nucleoside analogsimilar to zidovudine, a drug shown to cause a concentration-dependentdown-regulation of the erythropoietin receptor. When administered toHIV-infected individuals to treat zidovudine-associated anemia, Epoetinalpha may compensate for erythropoietin receptor down-regulation,thereby reversing the anemia.

In 13 out of the 18 individuals who received Epoetin alpha, the dose ofRBV was also reduced. Eight subjects had the RBV dose reduced prior toan initiation of Epoetin alpha. The RBV dose reduction may havepartially corrected the anemia (possibly through a decrease inhemolysis) while exogenous erythropoietin may have been required toovercome the reduced responsiveness of the bone marrow to Erythropoetin.

The administration of Epoetin alpha in IFN-α/RBV treated, HCV-infectedindividuals with profound decreases in hemoglobin provides importantclinical benefits by reversing the anemia, relieving anemia-associatedsymptoms such as fatigue and dyspnea, and improving an individual'sability to tolerate the full course of antiviral therapy at efficaciousdosages.

The present invention directs to an administering of EPO, e.g., aspecific predetermined amount of EPO, such as in a liquid preparationand/or as a vector in a liquid, to patients on a weekly basis. Theliquid preparation can contain EPO as disclosed in U.S. Pat. Nos.5,955,422; 5,756,349; 5,621,080; 5,618,698; 5,547,933; 4,703,008;5,856,298; 5,661,125; 5,106,760; 4,703,008; 5,856,298; 5,661,125;5,106,760; 4,558,006; 5,574,018; 5,354,934; 5,013,718; and 4,667,016.Alternatively, EPO can be administered as a vector that expresses theEPO in vivo, and this can reduce the frequency of the injection. Forexample, EPO can be administered as a vector, as a plasmid vector, or asa virus vector, such as a DNA virus or a retrovirus that expresses theEPO.

Compositions of the present invention can include liquid preparationsfor orifice, e.g., oral, nasal, anal, vaginal, peroral, intragastric,mucosal (e.g., perlingual, alveolar, gingival, olfactory or respiratorymucosa), etc.; administration such as suspensions, syrups or elixirs;and preparation for parenteral, subcutaneous, intradermal, intramuscularor intravenous administration (e.g., injectable administration), such assterile suspensions or emulsions. Such compositions may be in admixturewith a suitable carrier, diluent, or axcipient, such as sterile water,physiological saline, glucose or the like. The compositions can also belyophilized. The compositions can contain auxiliary substances, such aswetting or emulsifying agents, pH buffer agents, gelling or viscosityenhancing additives, preservatives, flavoring agents, colors, and thelike, depending upon the route of administration and the preparationdesired. Standard texts, such as “REMINGTON's PHARMACEUTICAL SCIENCE”,17^(th) Edition, 1985, incorporated herein by reference, may beconsulted to prepare suitable preparations, without undueexperimentation.

EPO is commonly administered when the hemoglobin level falls to a levelof 10 or 15 grams/dl. When EPO is administered to a patientsubcutaneously, it is given at a dose of about 40,000 units per week. Ifan inadequate response is seen, the dose can be increased to about60,000 units, or lowered to about 20,000 units on a weekly basisdepending on the response generated in a patient. EPO treatment for theIFN-α/RBV induced anemia is used as long as is necessary duringIFN-α/RBV combined treatment, which is usually about 12 months forgenotype 1, and may be about 6 months for hepatitis C genotype 2 or 3.

The invention will now be further described by the followingnon-limiting Examples, provided for illustration.

EXAMPLES Example 1

In an open-label, prospective pilot study, 56 subjects with chronic HCVinfection were treated with both RBV and IFN-α. Eighteen subjects withanemia (hemoglobin 10 g/dl or decreased g/dl) and reduced exercisetolerance following IFN-α/RBV initiation were treated with Epoetin alpha(up to 40,000 units per week subcutaneously). Remaining nonanemicsubjects (38/56) who did not receive Epoetin alpha served as a controlgroup. The two groups were followed for means of 25.3 and 21.2 weeks,respectively. At baseline, mean hemoglobin levels were not significantlydifferent in the subjects who received epoetin alpha compared to thosewho did not receive the medication (14.3 g/dl vs. 15.1 g/dL, n.s.). Atthe time of epoetin alpha initiation, the mean hemoglobin level declined25.4% (to 10.6 g/dL) in the group receiving this treatment. At the laststudy visit, mean hemoglobin levels were 12.7 g/dL among patientsreceiving epoetin alpha vs. 13.0 g/dL among controls (difference n.s.)reflecting a recovery of 65.5% in mean hemoglobin level in the epoetinalpha treated group. Two thirds of subjects receiving epoetin alphareported improved symptoms. This pilot study suggests that epoetin alphaincreases hemoglobin levels and improves dyspnea in subjects who developanemia after initiation of IFN-α and RBV.

Example 2

A study of interferon alone and interferon plus ribavirin for hepatitisC in HIV-infected patients was performed. Ten patients receivedinterferon alone and 11 received the combination treatment of interferonalpha and ribavirin. Median liver biopsy fibrosis score for both groupswas 2.1. In the interferon alone group, at 3 months, there was no changein HCV RNA, CD4 dropped from a median of 190 to 186 cells/ml and HIV RNAdropped from a median of 1500 copies per ml to less than 400. At thesame time point, 3 months, in the combination group, the HCV RNA droppedfrom 3.2 million to less than about 600 copies/ml. CD4 cells droppedfrom a median of 544 to 237, but percentage CD4 cells did not change.The main adverse event was anemia which occurred in 5/21(23.8%). Thiswas treated with erythropoietin 40,000 units weekly when the hemoglobindeclined to 10 g/dl. This treatment resulted in a rise in hemoglobin to12.7 g/dl after a median of 4 weeks treatment. This was the first reportof this treatment in patients with HIV infection as well as hepatitis C.

Subsequently 40,000 units of Erythropoetin alpha was used on a weeklybasis in patients with hemoglogin level of 11 gr/dl or less as a resultof ribavirin-induced anemia. This was done in both HIV seronegativeindividuals and HIV seropositive individuals. This has resulted in asubstantial increase in their quality of life as well as an increase inthe amount of ribavirin that they have been able to take.

Example 3

Fifty-six chronically HCV-infected (≥90 days) subjects who were treatedwith IFN-α (3 million units TIW) and RBV (1000 mg/d for subjects <75 kgor 1200 mg/d for subjects ≥75 kg) were entered into an open-label,pilot, prospective study at several centers. Subjects were excluded ifthey were infected with the human immunodeficiency virus type-1 or had ahistory of anemia, chronic renal disease, or coronary artery disease.RBV doses were adjusted by treating physicians as clinically indicatedthroughout the study.

Following INF-α/RBV initiation, subjects with new-onset anemia(hemoglobin ≤10 g/dL or ≥2 g/dL decrease in hemoglobin) or a decrease inhemoglobin accompanied by decreased exercise tolerance (providers'impressions) received epoetin alpha (up to 40,000 units per week,subcutaneously). Subjects who did not become anemic and who did notreceive epoetin alpha served as a control group.

After initiation of epoetin alpha therapy, subjects were evaluated at a2-week interval for 8 weeks and every 4 weeks thereafter for a maximumof 40 weeks. At each visit, laboratory safety parameters were obtainedand, for most subjects, health-care providers recorded whetherimprovement had occurred in symptoms that typically occur as aside-effect of anemia or after therapy with IFN-α/RBV (e.g., flu-likesymptoms, fatigue, dyspnea).

Hemoglobin levels were compared at baseline (prior to IFN-α/RBVinitiation) and at the last visit (IFN-α/RBV discontinuation visit)between subjects who received epoetin alpha and those who did not. Inaddition, clinical benefit of epoetin alpha therapy was evaluated bycomparing the percent decline in hemoglobin levels from baseline toinitiation of epoetin alpha with the percent recovery in these levels bythe last visit.

Fisher's exact test or the t-test was used to evaluate the significanceof differences between groups with respect to demographiccharacteristics. The t-test was used to compare mean hemoglobin levelsbetween individuals who received epoetin alpha and those who did not.

After a mean of 15.9±11.5 weeks (range: 3-44 weeks) following IFN-α/RBVtreatment initiation, 18 subjects (32.1%) who developed new-onset anemiaand reduced exercise tolerance were treated with epoetin alpha (up to40,000 units per week, subcutaneously). The comparison group comprised38 subjects (67.9%) who did not develop anemia and who did not receiveepoetin alpha.

Subjects in the group that received epoetin alpha and those in thecontrol group were similar with respect to age, the dose of RBV that wasprescribed, HCV genotype, and baseline hemoglobin (14.3±1.4 g/dL vs.15.1±1.4 g/dL, respectively; n.s.) (Table 1). Males were somewhatunderrepresented in the subjects who received epoetin alpha. In theepoetin alpha group, RBV doses were lowered prior to the initiation ofepoetin alpha therapy in eight subjects and at the time epoetin alphawas initiated in five additional subjects.

Subjects who received epoetin alpha were followed for a mean of25.3±15.6 weeks (range: 14-40 weeks) while individuals who did notreceive epoetin alpha were followed for a mean of 21.2±13.2 weeks(range: 2-47 weeks). The mean hemoglobin level in the subjects whoreceived epoetin alpha decreased from baseline by 25.4±8.9 percent (from14.3±1.4 g/dL to 10.6±1.0 g/dL). After initiation of epoetin alpha,hemoglobin increased by an average of 65.5±56.3 percent. At the lastvisit, mean hemoglobin levels were not significantly different in thetwo groups (12.7±1.7 g/dL in the epoetin alpha group vs. 13.1±1.4 g/dLin the comparison group; n.s.).

Among the 53 patients with complete data, the frequency and duration ofsymptoms were similar between the two groups with two exceptions (Table2). Dyspnea was reported by significantly more subjects in the epoetinalpha group than in the group that did not receive epoetin alpha (80.0%vs. 23.6%, respectively, P<0.001). Likewise, the median duration offatigue was significantly more prolonged among those in the epoetinalpha group in comparison to those in the other group (8.2 vs. 5.3weeks, respectively, P<0.05).

Anecdotal reports by health care providers indicated that anemia-relatedsymptoms improved in two-thirds of patients in the epoetin alpha groupby the time of the last visit.

Table 3 shows a lab average for HCV/HIV patients on Interferon and RBV.

TABLE 1 PATIENT PROFILE Epoetin alpha No epoetin alpha (N = 18) (N = 38)Mean (±s.d.) age (yrs) 47.7 (7.8) 44.5 (7.4) Male (%) 38.9 68.4 Mean(±s.d.) baseline 14.3 (1.4) 15.1 (1.4) hemoglobin (g/dL) RBV dose (%):1000 mg/d 72.2 84.2 1200 mg/d 27.8 15.8 HCV genotype (%)*: 1a or 1b 53.355.3 Other 33.3 23.7 Untested 13.3 21.0 *Missing data for three patientsin the Epoetin alpha group.

TABLE 2 SUBJECTS' SYMPTOMS Epoetin alpha No epoetin alpha (N = 15) (N =38) Dyspnea (%) 80.0 23.6* Median duration (wks) 6.6 4.6 Fatigue (%)73.3 60.5 Median duration (wks) 8.2 5.3^(†) Flu-like symptoms 46.7 15.8Median duration (wks) 2.7 5.4 *P < .001 ^(†)P < .05

TABLE 3 LAB AVERAGES FOR HCV/HIV PATIENTS ON INTERFERON AND RBA TotalInitials Testosterone Triglycerides Cholesterol LDL HDL Glucose ALT ASTProtein Ablumin Epo ANC Hgb WBC RR 1077.3 99.1 149 89.8 39.5 131.5 36.958.4 7.5 4 901.6 12.9 2.7 AZ 372.7 176.2 89 38.1 23.2 78 192.8 131.8 6.73.6 17.7 5793.3 12.1 6.8 PB 955.3 247.2 160.7 90.8 43.6 70 42.7 34.4 8.14.5 2562 12.2 4.3 FK 93.6 171.3 108 45 86.8 33.3 51.3 7.6 3.8 3057.710.9 6.2 SSC 544 145.5 129 75.8 24 95 86 60.1 7.6 4.4 AC 1140 149.5184.5 116.5 38 103 37 22 7 4.6 DT 1461.3 260.7 258.7 108.6 47.4 86.593.3 71.5 8.3 4 10.6 888.6 14.2 4.7 JC 413 171.5 157 98 24.5 91.5 26.528 7.7 3.7 1773 14.7 3.4 FG 101 172 119 27.5 101 138.5 93.5 7.4 4.1 RA583.6 168.6 121.8 75 25 104 52.2 37.3 6.8 4.3 2669.8 14.2 5.2 RV 209.8247.8 238.8 161.4 27.7 71.6 54 50 8.3 4.2 59.2 2291.5 10 5.1 RK 590.8135 207.6 135.8 36.2 93.3 23 37.8 8.4 4.2 1914 12.9 3.4 CL 1333.3 289.8166.7 97.9 30.2 95.9 43.3 40.8 7 4.2 2084.5 14.1 4.6 JS 476.8 364.3129.8 56.2 13.1 103.7 126 120.4 7.7 4.1 1924.7 10.6 3.1 PR 18.3 85.7153.1 90.6 45.5 94.6 70.6 118.2 7.4 3.8 3470 12.4 5.1 RA 1258 105 186.5140 25.5 85.5 169.7 77.3 7.2 4 2333.3 16.1 4.4 HD 1635.9 111 154.3 7562.9 86.2 62.7 70.2 8.4 4.1 2160.9 13.9 4 JF 908.3 384.3 273.5 157.524.9 81.7 118 96.7 8.2 4.6 1931.6 14.6 4.5 LM 696.5 172.1 167.7 101.692.2 90.2 46 41.8 7.6 4.3 9 1590.2 12.4 4.7 RC 17.3 89.2 193.1 108.756.4 101.5 49.1 56.9 7.2 4 9.4 1828.9 13.2 4.3 JE 599.7 150 186 124.331.1 84.3 148.8 83.8 7.7 4.3 1244.6 14.5 2.9 MP 451 213.7 186.1 108.135.2 101.9 67.3 40 7.2 4.1 1275.4 14.4 2.6 MR 15 373 130.2 65.5 14.1108.4 44.3 70.1 6.3 2.4 69.2 1452.5 11.3 3.3 MEAN 719.3 188.4 172.5104.7 36.8 93 76.6 64.9 7.5 4.1 29.2 2157.4 13.1 4.265 MEDIAN 595.25168.6 167.7 104.8 33.15 93.3 54 58.4 7.6 4.1 14.15 1928.15 13.2 4.35

Example 4

Once-weekly recombinant human erythropoietin (epoetin alpha) facilitatesoptimal ribavirin (RBV) dosing in Hepatitis C virus (HCV)-infectedpatients receiving Interferon-α-2b (IFN)/RBV combination therapy. RBVdose reduction or discontinuation has been the standard approach totreating hemoglobin (Hb) decreases resulting from IFN/RBV therapy inHCV-infected patients. A recent retrospective analysis suggested thatmaintaining RBV doses greater than 80% (10.6 mg/kg per day) of therecommended dose was associated with higher sustained virologic responserates (McHutchison et al., AASLD. 2000:223 A. Abstract 247). Anopen-label, randomized, multicenter study was conducted to assess theefficacy and tolerability of once-weekly (QW) epoetin alpha therapy forhemoglobin decreases associated with IFN/RBV therapy and to evaluate theimpact of epoetin alpha therapy on RBV dosing.

HCV-infected patients who developed hemoglobin levels ≤12 g/dL duringthe first 24 weeks of combination IFN/RBV therapy (n=60) were randomlyassigned to treatment with epoetin alpha (40000 units) subcutaneouslyonce weekly for up to 36 weeks or standard of care (SOC). The primaryand secondary efficacy end points were changes from baseline (at Week16) in hemoglobin level and RBV dose, respectively.

At baseline, mean hemoglobin levels and RBV doses were similar betweenthe epoetin alpha and SOC groups (11.0 g/dL, for both, and 998 vs. 986mg/day, respectively). At the last available assessment (LAST), the meanchanges from baseline hemoglobin levels were +2.7 vs. +0.4 g/dL forepoetin alpha vs. SOC groups (P<0.05), respectively; and the meanchanges in RBV doses were −25 and −190 mg/day, respectively (P<0.05).The last available assessment mean hemoglobin level in the epoetin alphagroup (13.8 g/dL) was significantly greater (P<0.05) than that of theSOC group (11.3 g/dL). Based on the last available assessment, 77% vs.50% (P<0.01) of patients in the epoetin alpha vs. SOC groups,respectively, were treated with RBV doses greater than 10.6 mg/kg perday.

Once weekly epoetin alpha therapy effectively increased hemoglobinlevels in HCV-infected patients treated with INF/RBV. Epoetin alpha waswell tolerated and increased the proportion of patients able to tolerateRBV doses greater than 10.6 mg/kg per day, which has been shown toincrease the likelihood of sustained virologic response to IFN/RBVtreatment.

Attached as FIG. 2 are plots of hemoglobin levels by time for 18EPO-treated patients.

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What is claimed is:
 1. A method for treating hepatitis C in a HIVnegative patient in need thereof comprising administering ribavirin(RBV) or RBV and interferon-alpha (IFN-alpha), monitoring hemoglobinlevel of the HIV negative patient who has been administered RBV or RBVand IFN-alpha, initiating administration of EPO when the hemoglobinlevel of the patient is greater than 10 gm/dL but no greater than 12gm/dL, wherein Erythropoetin (EPO) is administered to the patient in anamount effective to reduce the effects of IFN-alpha/RBV induced anemia,at a weekly dose of about 10,000 to 70,000 units, wherein the RBV isadministrated at a level of greater than 10.6 mg/kg per day.
 2. Themethod of claim 1 wherein the hepatitis C is genotype 2 and/or 3 and/or1 and/or
 4. 3. The method for treating hepatitis C comprisingadministering ribavirin and interferon-alpha wherein the improvement asclaimed in claim 1 comprises administering to patients subcutaneously ata weekly dose of about 20,000 to 60,000 units of erythropoietin.
 4. Themethod of claim 1 wherein the ribavirin is administered in a dose ofabout 800 to 1200 mg/day.
 5. The method of claim 1, wherein EPO isadministered to the patient subcutaneously for at least about sixmonths.